The present invention resides generally in the field of regenerative medicine using hematopoietic stem and progenitor cells. More particularly, the present invention relates to hematopoietic reconstitution and replacement in patients with various diseases and metabolic disorders. Regenerative medicine is a newly evolving method of targeted treatments of a large number of life threatening diseases. It involves three therapeutic technologies—cellular therapy, cell engineering and gene therapy—all three involved in the utilization of human tissues.
Cellular therapy has the broadest therapeutic applications in various diseases such as immunodeficiencies, hemoglobinopathies, metabolic disorders, diseases of the nervous system and some malignancies.
Cell engineering is another promising area of novel therapy that may have a major impact on management of cardiac ischemic diseases, treatment of bone and joint diseases, and chronic wounds with bone marrow or mesenchymal stem cells.
Gene therapy is another form of technology that has held considerable promise although unexpected long term effects of gene therapy, such as malignancies and death have limited its clinical application.
Cellular therapies as well as cell engineering, the most important technologies in regenerative medicine, require a large supply of stem cells. At present there is an insufficient supply of human stem cells for current applications and this lack will be compounded as the regenerative medicine reaches its full potential. The current sources of human hematopoietic stem cells include adult peripheral blood, cord blood, and the adult bone marrow. The morphologically recognizable and functionally capable cells circulating in blood include erythrocytes, neutrophilic, eosinophilic, and basophilic granulocytes, B-, T-, nonB-, non T-lymphocytes, and platelets. These mature cells derive from and are replaced, on demand, by morphologically recognizable dividing precursor cells for the respective lineages such as erythroblasts for the erythrocyte series, myeloblasts, promyelocytes and myelocytes for the granulocyte series, and megakaryocytes for the platelets. The precursor cells derive from more primitive cells that can simplistically be divided into two major subgroups: stem cells and progenitor cells. The definitions of stem and progenitor cells are operational and depend on functional, rather than on morphological, criteria. Stem cells have extensive self-renewal or self-maintenance capacity, a necessity since absence or depletion of these cells could result in the complete depletion of one or more cell lineages, events that would lead within a short time to disease and death. Some of the stem cells differentiate upon need, but some stem cells or their daughter cells produce other stem cells to maintain the precious pool of these cells. Thus, in addition to maintaining their own kind, pluripotent stem cells are capable of differentiating into several sublines of progenitor cells with more limited or no self-renewal capacity or no self-renewal capacity. These progenitor cells ultimately give rise to the morphologically recognizable precursor cells. The progenitor cells are capable of proliferating and differentiating along one, or more than one, of the myeloid differentiation pathways.
Under the appropriate growth conditions, the stem or progenitor cells will go through a sequence of proliferation and differentiation yielding mature end stage progeny, which thus allows the determination of the cell type giving rise to the colony. If the colony contains granulocytes, macrophages, erythrocytes, and megakaryocytes (the precursors to platelets), then the cells giving rise to them would have been pluripotent cells. To determine if these cells have self-renewal capacities, or stemness, and can thus produce more of their own kind, cells from these colonies can be replated in vivo or in vitro. Those colonies, which upon replating into secondary culture plates, give rise to more colonies containing cells of multilineages, would have contained cells with some degree of stemness. The stem cell and progenitor cell compartments are themselves heterogeneous with varying degrees of self-renewal or proliferative capacities. Self-renewal would appear to be greater in those stem cells with the shortest history of cell division, and this self-renewal would become progressively more limited with subsequent division of the cells.
Reconstitution of the hematopoietic system has been accomplished by bone marrow transplantation. The stem and progenitor cells in donated bone marrow can multiply and replace the blood cells responsible for protective immunity, tissue repair, clotting, and other functions of the blood. In successful bone marrow transplantation, the blood, bone marrow, spleen, thymus and other organs of immunity are repopulated with cells derived from the donor.
Cryopreservation of Cells—Freezing and thawing are destructive to most living cells. Upon cooling, as the external medium freezes, cells equilibrate by losing water, thus increasing intracellular solute concentration. Below about −10-15° C., intracellular freezing will occur. Both intracellular freezing and solution effects are responsible for cell injury. It has been proposed that freezing destruction from extracellular ice is essentially a plasma membrane injury resulting from osmotic dehydration of the cell. Cryoprotective agents and optimal cooling rates can protect against cell injury. Cryoprotection by solute addition is thought to occur by two potential mechanisms: by penetration into the cell, reducing the amount of ice formed; or kinetically, by decreasing the rate of water flow out of the cell in response to a decreased vapor pressure of external ice. Different optimal cooling rates have been described for different cells. Various groups have looked at the effect of cooling velocity or cryopreservatives upon the survival or transplantation efficiency of frozen bone marrow cells or red blood cells. The successful recovery of human bone marrow cells after long-term storage in liquid nitrogen has been described (1983, American Type Culture Collection, Quarterly Newsletter 3(4):1). There are many methods of cryopreservation and many cryoprotective cocktails have been proposed. However, the recovery of viable cells is still very poor (generally not better than 60%) (Hunt et al., Cryo Letters 2006, 27: 73-86). Experience with standard protocols based on those designed for cord blood stem cells have clearly indicated that there is need for further improvement in the procedures for cryopreservation. Moreover, it is known that standard freezing solutions that contain 10% dimethyl sulfoxide (DMSO), which routinely give 60% cell recovery after thawing, can induce neuro- and cardio-toxic reactions. Consequently the present invention fucusses effort in this area on minimizing the toxic effects of DMSO by radically decreasing its concentration in the freezing cocktail.
Gene Therapy refers to the transfer and stable insertion of new genetic information into cells for the therapeutic treatment of diseases or disorders. The foreign gene is transferred into a cell that proliferates to spread the new gene throughout the cell population. Thus stem cells, or pluripotent progenitor cells, are usually the target of gene transfer, since they are proliferative cells that produce various progeny lineages which will potentially express the foreign gene. Most studies in gene therapy have focused on the use of hematopoietic stem cells. High efficiency gene transfer systems for hematopoietic progenitor cell transformation have been investigated for use. Reports on the development of viral vector systems indicate a higher efficiency of transformation than DNA-mediated gene transfer procedures (e.g., Ca3PO4)2 precipitation and DEAE dextran) and show the capability of integrating transferred genes stably in a wide variety of cell types. Recombinant retrovirus vectors have been widely used experimentally to transduce hematopoietic stem and progenitor cells. Genes that have been successfully expressed in mice after transfer by retrovirus vectors include human hypoxanthine phosphoribosyl transferase. Bacterial genes have also been transferred into mammalian cells, in the form of bacterial drug resistance gene transfers in experimental models. Introduction of drug resistance genes into hematopoietic stem cells has been accomplished using a retroviral vector system. Adenovirus vectors have been used successfully to transduce mammalian cell lines to neomycin resistance. Other viral vector systems that have been investigated for use in gene transfer include parvoviruses and vaccinia viruses. Other methods of gene transfer including microinjection, electroporation, liposomes, chromosome transfer, and transfection techniques have been published in literature and are incorporated herein.
The promise of stem cell therapy through cellular, cell engineering and/or gene therapies, has generated tremendous hope for the development of new sources of embryonic stem cells, including cloning and creation of new embryonic stem cell lines. However, there are still very significant problems to overcome before embryonic stem cells can be utilized in therapy. Likewise the use of fetal stem cells derived from elective abortions is plagued with many problems. Most elective abortions occur early in gestation hence there are little or no hematopoeitic cells in long bones because they have still not translocated to their ultimate destination from the liver. Moreover, there are significant moral and ethical problems in using fetal tissue derived from elective abortions. The present invention has overcome some of these problems by establishing fetal stem cells from second trimester miscarriages. The second trimester fetal tissue has many advantages. The fetal tissue, especially the hematopoietic tissue, has the optimal characteristics for long term engraftment and regenerative properties. This is related to a very high number of transplantable primitive cells with high clonogenic and proliferative properties. Furthermore, in contrast to other sources of stem cells described in the prior art (U.S. Pat. No. 5,004,681 to E. A. Boyse et al; U.S. Pat. No. 5,192,553 to E. A. Boyse et al; U.S. Pat. No. 6,143,289 to Hal E. Broxmeyer et al; U.S. Pat. No. 6,461,645 E. A. Boyse et al; U.S. Pat. No. 6,613,568 to D. S. Kaufman et al; U.S. Pat. No. 6,887,706 to S-C Zhang et al; U.S. Pat. Nos. 6,987,102; 7,005,252 to James A Thomson; and U.S. Pat. No. 7,029,913 to James A. Thomson), the fetal cells in the present invention have very low immunogenicity because of the age of the specimen (16-20 weeks), where the immune system is still poorly developed. The therapeutic mechanism of the fetal stems of the invention, though not fully understood, may be related to cellular reconstitution, replacement of detected cells or enhancement of their function by release of endogenous trophic factors. Tissue derived from second trimester lost pregnancies is completely free from the moral and bioethical problems associated with other fetal or embryonic sources of stem cells because it is considered cadaveric tissue and is fully acceptable for use in therapy.